Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds - PubMed (original) (raw)

Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds

Jie Lu et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Seed size is important to crop domestication and natural selection and is affected by the balance of maternal and paternal genomes in endosperm. Endosperm, like placenta in mammals, provides reserves to the developing embryo. Interploidy crosses disrupt the genome balance in endosperm and alter seed size. Specifically, paternal-excess crosses (2 × 4) delay endosperm cellularization (EC) and produce larger seeds, whereas maternal-excess crosses (4 × 2) promote precocious EC and produce smaller seeds. The mechanisms for responding to the parental genome dosage imbalance and for gene expression changes in endosperm are unknown. In plants, RNA polymerase IV (PolIV or p4) encoded by NRPD1a is required for biogenesis of a major class of 24-nt small interfering RNAs (also known as p4-siRNAs), which are predominately expressed in developing endosperm. Here we show that p4-siRNA accumulation depends on the maternal genome dosage, and maternal p4-siRNAs target transposable elements (TEs) and TE-associated genes (TAGs) in seeds. The p4-siRNAs correlate negatively with expression levels of AGAMOUS-LIKE (AGL) genes in endosperm of interploidy crosses. Moreover, disruption of maternal NRPD1a expression is associated with p4-siRNA reduction and AGL up-regulation in endosperm of reciprocal crosses. This is unique genetic evidence for maternal siRNAs in response to parental genome imbalance and in control of transposons and gene expression during endosperm development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Seed morphology and chromosome counts in interploidy crosses. (A) Seed size and morphology in diploids, tetraploids, and triploids (2 × 4 or 4 × 2) in A. thaliana C24. m, maternal; p, paternal. By convention, the maternal parent is listed first in a genetic cross. (B) Chromosome counts in diploid, triploid, and tetraploid flowers in A. thaliana Col-0. (C) Seed weight in interploidy crosses. (D) Developing seeds dissected at 3–7 d after pollination (DAP) in Col-0.

Fig. 2.

Fig. 2.

Small RNA distribution in interploidy crosses. (A) Size distribution of 20–25 nt small RNA reads in Col-0 seeds of 2 × 2 (blue), 4 × 4 (green), 2 × 4 (cyan), and 4 × 2 (magenta). (B) Distribution of 20–25 nt small RNAs in genes, TEs, miRNA, and ta-siRNA targets, and intergenic regions. (C and D) 24-nt small RNA densities (100-bp sliding window) in 5′ upstream (2 kb), transcribed, and 3′ downstream (2 kb) regions of TE genes in seeds (C) and leaves (D). (E) Small RNA blot analysis of miR172, miR832, and miR396 in diploids, triploids, and tetrpaloids at 6 DAP.

Fig. 3.

Fig. 3.

Distribution of TE-associated genes (_TAG_s) and parent-of-origin effects of siRNAs on TAG expression in reciprocal interploidy crosses. (A) Proportions of _TAG_s and locations of TEs (triangles) in coding sequences (gray box) and within 2 kb up or downstream of 5′ and 3′ regions (extended lines) in A. thaliana Col-0. (B and C) Small RNA densities (100-bp sliding window) in 5′ upstream (2 kb), transcribed, and 3′ downstream (2 kb) regions of _TAG_s and non-_TAG_s in seeds (B) and leaves (C). (D and E) Percentage of up-regulated (red) and down-regulated (blue) _TAG_s or non-_TAG_s in 2 × 4 vs. 4 × 2 crosses (D) and in 2 × 6 vs. 6 × 2 crosses (E). (F and G) Heat maps of gene expression changes in endosperm transcription factor (EFT) genes (n = 27) (F) and silique transcription factor (STF) genes (n = 25) (G) in two replicated experiments (Reps. 1 and 2); color bar indicates up (red) and down (green) regulation; black dots indicate up-regulated genes at statistically significant levels between 2 × 4 and 4 × 2 endosperm.

Fig. 4.

Fig. 4.

Maternal siRNAs are associated with expression of AGL genes and FWA. (A_–_H) siRNA hotspots (Left) and qRT-PCR analysis (Right) of AGL28 (A), AGL36 (B), AGL40 (C), AGL62 (D), AGL87 (E), AGL90 (F), AGL91 (G), and FWA (H) in 2 × 4 and 4 × 2 triploids and their parents (2 × 2 and 4 × 4). Diff., siRNA differences between 4 × 2 and 2 × 4; positive, Above the line; negative, Below the line; gray box, gene; black box, transposon. Genomic coordinates are shown Above each diagram, and SEs were calculated from three biological replicates.

Fig. 5.

Fig. 5.

siRNA production and AGL expression are dependent on RNA polymerase IV (NRPD1A) in endosperm and a model for endosperm development in interploidy crosses. (A) qRT-PCR analysis (relative expression levels, REL) of NRPD1a expression in endosperm of interploidy crosses. (B_–_D) qRT-PCR analyses of AGL62 (B), AGL91 (C), and AGL40 (D) expression (n = 3 biological replicates). (E) Small RNA blot analysis of p4-siRNA (siR02), _AGL40_-siRNA, and _AGL91_-siRNA in endosperm of interploidy crosses (n = 2 biological replicates). (Left) _AGL91_-siRNAs were present in seeds but not in siliques. miR166 was used as a control. (F) Model for the role of maternal siRNA-mediated AGL expression in endosperm and seed development (see text for explanation). Multiple dots and an elongated black rod in each diagram represent the endosperm and embryo cells, respectively.

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